Modified retroviral vectors

Abstract

The present invention relates to methods and compositions for the elucidation of mammalian gene function. Specifically, the present invention relates to methods and compositions for improved mammalian complementation screening, functional inactivation of specific essential or non-essential mammalian genes, and identification of mammalian genes which are modulated in response to specific stimuli. In particular, the compositions of the present invention include, but are not limited to, replication-deficient retroviral vectors, libraries comprising such vectors, retroviral particles produced by such vectors in conjunction with retroviral packaging cell lines, integrated provirus sequences derived from the retroviral particles of the invention and circularized provirus sequences which have been excised from the integrated provirus sequences of the invention. The compositions of the present invention further include novel retroviral packaging cell lines.

What is claimed is: 
     
       1. A retroviral vector comprising: (a) a 3' LTR sequence for integration of the vector into chromosomal DNA of a host cell;   (b) a heterologous nucleic acid sequence to be transcribed in the host cell, or one or more restriction cloning sites for cloning the heterologous nucleic acid sequence into the vector;   (c) proviral excision elements, contained within the 3' LTR sequence, for excising a proviral form of the vector from chromosomal DNA.   
     
     
       2. The vector of claim 1, wherein said heterologous nucleic acid sequence comprises a cDNA or gDNA coding sequence for a polypeptide. 
     
     
       3. A retroviral library comprising a multiplicity of the retroviral vectors of claim 1, having different heterologous sequences. 
     
     
       4. A retroviral particle including the retroviral vector of claim 1. 
     
     
       5. An integrated provirus derived from the retrovirus of claim 1, said provirus integrated into cells isolated in culture. 
     
     
       6. An excised provirus obtained by the excision of a provirus integrated by the vector of claim 1. 
     
     
       7. The vector of claim 1 wherein said vector is a replication-deficient virus. 
     
     
       8. The vector of claim 1, wherein said vector further includes a proviral recovery element for isolating the vector from a mixture of nucleic acids. 
     
     
       9. The vector of claim 1, wherein said excision elements comprise enzyme-assisted site-specific integration sequences. 
     
     
       10. The vector of claim 9, wherein said excision elements include recombinase target sites. 
     
     
       11. The vector of claim 10, wherein said recombinase target sites are target sites for Cre recombinase or Flp recombinase. 
     
     
       12. The vector of claim 1, wherein said excision elements include restriction enzyme sites. 
     
     
       13. The vector of claim 1, wherein said excision elements are positioned in the vector such that, upon excision of the vector from chromosomal DNA, the excised vector can be used directly to generate virus for subsequent rounds of infection. 
     
     
       14. The vector of claim 1, wherein said vector further includes a packaging signal for packaging the vector in an infectious viral particle. 
     
     
       15. The vector of claim 1, further comprising a polycistronic message cassette for transcribing the heterologous nucleic acid sequence as a polycistronic message. 
     
     
       16. The vector of claim 15, wherein said polycistronic message cassette comprises the heterologous nucleic acid sequence, or the restriction cloning sites for cloning the heterologous nucleic acid sequence, disposed in said vector proximal to one or more marker genes such that the heterologous nucleic acid sequence and marker gene(s) are transcribed as a polycistronic message. 
     
     
       17. The vector of claim 15, wherein said polycistronic message includes internal ribosome entry sites (IRES) between coding sequences of the message. 
     
     
       18. The vector of claim 8, wherein said proviral recovery element comprises a nucleic acid sequence specifically bound by a DNA binding polypeptide. 
     
     
       19. The vector of claim 1, 25 or 28, further comprising at least one bacterial origin of replication disposed in the vector such that, upon excision, the origin of replication is present in the provirus. 
     
     
       20. The vector of claim 19, wherein said bacterial origin of replication is a non-pUC ori. 
     
     
       21. The vector of claim 19, wherein said bacterial origin of replication is a single-stranded origin of replication. 
     
     
       22. The vector of claim 20, wherein said bacterial origin of replication is selected from the group consisting of RK2 OriV and f1 phage Ori. 
     
     
       23. The vector of claim 1, further comprising a selectable bacterial marker gene. 
     
     
       24. The vector of claim 23, wherein said selectable bacterial marker gene renders a bacterial host cell resistant to a drug or complements a cellular phenotype. 
     
     
       25. The vector of claim 23, wherein said selectable bacterial marker gene renders a bacterial host cell resistant to a drug selected from the group consisting of kanamycin/G418, zeocin, actinomycin, ampicillin, gentamycin, tetracycline, chloramphenicol and penicillin. 
     
     
       26. The vector of claim 1, further comprising a mammalian marker gene, the expression of which provides a detectable phenotype in a host cell. 
     
     
       27. The vector of claim 26, wherein expression of said mammalian marker gene renders the host cell resistant to a drug or complements a cellular phenotype. 
     
     
       28. The vector of claim 26, wherein said mammalian marker gene encodes a protein providing resistance to kanamycin/G418, hygromycin, mycophenolic acid or neomycin. 
     
     
       29. The vector of claim 26, wherein said mammalian marker gene encodes a fluorescent protein, or an enzyme which can alter the fluorescence of the host cell. 
     
     
       30. The vector of claim 29, wherein said mammalian marker gene encodes a green fluorescent protein. 
     
     
       31. The vector of claim 1, further comprising a lethal stuffer fragment, the expression of which provides a detectable phenotype in the host cell, the expression of the lethal stuffer fragment being dependent on the presence or absence in the vector of the heterologous nucleic acid sequence. 
     
     
       32. The vector of claim 1, 10 or 18, wherein said heterologous nucleic acid sequence includes a coding sequence for a polypeptide. 
     
     
       33. The vector of claim 32, wherein said coding sequence encodes an intracellular polypeptide. 
     
     
       34. The vector of claim 32, wherein said coding sequence encodes a secreted or cell surface polypeptide. 
     
     
       35. The vector of claim 1, wherein said heterologous nucleic acid sequence includes a genetic suppressor element. 
     
     
       36. The vector of claim 35, wherein said genetic suppressor element is selected from the group consisting of an antisense construct, a coding sequence for a dominant negative mutant or fragment of protein, and a ribozyme. 
     
     
       37. The vector of claim 1, further comprising a constitutive transcriptional regulatory sequence for regulating transcription of the heterologous nucleic acid in the host cell. 
     
     
       38. The vector of claim 1, further comprising an inducible transcriptional regulatory sequence for regulating transcription of the heterologous nucleic acid in the host cell. 
     
     
       39. The vector of claim 1, wherein said vector is incorporated in an artificial chromosome. 
     
     
       40. The vector of claim 1, wherein said retroviral vector is derived from a replication-deficient retrovirus lacking all or a portion of the retroviral gag, pol and/or env genes. 
     
     
       41. The vector of claim 1, wherein said retroviral vector is derived from pBABE. 
     
     
       42. The vector of claim 1, wherein said LTR sequences include said excision elements, and said excised vector can be used directly to generate packaged retroviral vectors. 
     
     
       43. The vector of claim 42, wherein said vector includes a self-inactivating LTR. 
     
     
       44. The vector of claim 1, wherein said vector is a closed circular nucleic acid. 
     
     
       45. The vector of claim 1, wherein said vector is a linear nucleic acid. 
     
     
       46. The vector of claim 1, wherein said vector is DNA. 
     
     
       47. The vector of claim 1, wherein said vector is DNA. 
     
     
       48. The vector of claim 1, wherein said vector is packaged in a viral particle. 
     
     
       49. A library of vectors comprising a population of vectors according to claim 1, 11 or 13, the library including vectors having different heterologous nucleic acid sequences. 
     
     
       50. The vector library of claim 49, wherein said library comprises, as the heterologous nucleic acid sequences, a population of cDNA sequences. 
     
     
       51. The vector library of claim 50, wherein said cDNA library is a normalized cDNA library. 
     
     
       52. The vector library of claim 49, wherein said library comprises, as the heterologous nucleic acid sequences, a population of coding sequences for a peptide library. 
     
     
       53. The vector library of claim 52, wherein said peptide library is a constrained peptide library, as for example, part of a fusion protein. 
     
     
       54. The vector library of claim 49, wherein said library comprises, as the heterologous nucleic acid sequences, a population of genetic suppressor elements. 
     
     
       55. The vector library of claim 54, wherein said genetic suppressor elements are selected from the group consisting of an antisense constructs, coding sequences for fragments of proteins and ribozymes. 
     
     
       56. The vector of claim 8, wherein said provirus recovery element is a lac operator nucleic acid sequence, a tet operator nucleic acid sequence or a lambda operator nucleic acid sequence. 
     
     
       57. The vector of claim 18, wherein said DNA binding polypeptide is a lac repressor, a tet repressor or a lambda repressor. 
     
     
       58. The retroviral vector library of claim 3, wherein said cDNA library is a normalized cDNA library. 
     
     
       59. The retroviral library of claim 3, wherein said library is a single-stranded library. 
     
     
       60. The excised provirus of claim 6, wherein said excised provirus is circularized.